Mastitis is the most common and most costly disease of dairy animals in the world. Mastitis is diagnosed by observing heat, swelling, redness, pain and abnormal milk in the affected gland. Most dairy farmers treat this disease with antibiotics. Treatment is usually instituted immediately upon diagnosis of the disease. The antibiotic chosen for treatment is based upon past experience with what has been working pure guess work or the results of an antibiotic sensitivity test.
Farmers generally do not submit milk samples for antibiotic sensitivity tests because of the unacceptable lag time between the submission of milk samples and the receipt of the test results. That is, by the time the antibiotic sensitivity test results are reported to the farmer, i.e., 48 hours to 1 week, most cases of mastitis have either cleared up or progressed to a much greater severity. Some animals may even lose function of the affected quarter while others may die from septicemia. Thus antibiotic sensitivity tests are not generally employed.
The two types of antibiotic sensitivity tests which are currently employed are the disc sensitivity test (Bauer. A. W. et al, Am. J. Chem. Path., 45:493 (1966)) and the broth or agar dilution antibiotic susceptibility test (National Committee for Clinical Laboratory Standards. Vol. 5. No. 22 (1985)). To date, the disc sensitivity test is the most commonly employed antibiotic sensitivity test for mastitis.
In the disc sensitivity test, milk, collected using sterile techniques, is streaked out on a blood agar plate. Then, pathogenic microorganisms, i.e., Staphlococcus aureus, Streptococcus agalactiae Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli and Klebsiella species, which account for over 90% of bovine mastitis pathogens, are isolated after 12 to 24 hours of incubation on the blood agar plate. After isolation, a standard amount of the microorganisms, sometimes diluted in trypticase soy broth, is streaked on a Mueller Hinton agar plate and allowed to grow, thereby forming a carpet of microorganisms after 18 to 24 hours. Then, discs, which have previously been impregnated with various antibiotics, are placed in the plate. If the antibiotic is effective against the particular type of microorganism present. a ring or zone of inhibition appears on the plate. A sliding scale is used to measure how strongly the antibiotic inhibits the bacterial growth.
The disc sensitivity test is disadvantageous not only because of the length of time required to run the test, i.e., 36 to 48 hours but, also, because sometimes there is a poor correlation between the in vitro results and the in vivo response to a particular antibiotic. This is believed to be due to the fact that antibiotics behave differently in milk than in agar (Laboratory and Field Handbook on Bovine Mastitis, Eds. Research Committee of National Mastitis Council, National Mastitis Council Inc., Arlington, Va. (1987): and Owens, W. E. et al J. Dairy Sci., 70:1946 (1987)). In addition the disc sensitivity test is less accurate than the broth or agar dilution antibiotic sensitivity test (Turck M. et al. Ann. Int. Med., 58:56 (1963)).
The broth or agar dilution antibiotic sensitivity test, in its miniaturized form, is believed to more closely predict the effect of an antibiotic in vivo because it uses specific concentrations of antibiotics that correlate with known serum concentrations necessary to be effective against a mastitis causing microorganism infection. In spite of this, due to the expense, availability and lack of veterinary antibiotic panels, this test is rarely performed on milk.
In the broth or agar dilution antibiotic sensitivity test, a known quantity of microorganisms, usually isolated on a blood agar plate from the original sample, is placed in wells of a microtiter plate, a series of tubes or a commercially available cassette consisting of many wells. The quantity of microorganisms is measured by adding the microorganisms to culture medium until the turbidity matches that of a standard, i.e. the MacFarland Standard. The greater the turbidity the more microorganisms are present. The MacFarland Standard is employed because if there are not enough microorganisms, some wells will not receive any microorganisms and no growth would give false results. Further if too many microorganisms are present, they might overgrow and overwhelm the antibiotic, again giving false results.
The wells of the microtiter plate the series of test tubes or the commercially available cassette to which the microorganism-culture medium mixture is added, contain serial dilutions of various antibiotics. After 6 to 16 hours of incubation the wells, test tubes or cassette are observed for turbidity. High turbidity indicates growth and means the microorganisms are resistant to the antibiotic at that level. Low turbidity means the microorganisms are sensitive to the antibiotic at that level. Low turbidity in a well, test tube or cassette having a low concentration of antibiotic indicates high sensitivity. Antibiotic concentration can be correlated with serum levels achieved using typical therapeutic dosages. Thus, this test is more quantitative than the disc sensitivity test.
However, the broth or agar dilution antibiotic sensitivity test is disadvantageous not only because of the length of time required to run the test, i.e., 18 to 40 hours but, also, because this test is not carried out on the original sample of milk. i.e.. the original sample of milk is first cultured and the microorganisms identified. Further this test is carried out in a culture medium or laboratory broth rather than in milk. Antibiotics behave differently in milk than in culture media (Laboratory and Field Handbook on Bovine Mastitis, Eds. Research Committee of National Mastitis Council, National Mastitis Council, Inc., Arlington, Va. (1987): and Owens, W. E. et al, J. Dairy Sci., 70:1946 (1987)). In addition the currently available test procedure is too difficult and expensive to run in a veterinarian's office or on a farm.
Thus, to date, a cost effective, fast, and accurate test for determining the antibiotic sensitivity of the major pathogenic microorganisms present in milk obtained from mammals afflicted with mastitis has not been developed (Laboratory and Field Handbook on Bovine Mastitis, Eds. Research Committee of National Mastitis Council, National Mastitis Council, Inc., Arlington, Va. (1987)). Moreover, the method of the present invention is unique in that it tests for antibiotic sensitivity without the need for first identifying the microorganism.
The fermentation of lactose to lactic acid is common to many microorganisms. This fact is used by microbiologists as an aid in the identification of microorganisms. That is, an unknown microorganism is either a lactose fermenter or it is not.
The Hotis test, developed in 1936 (Hotis, R. P. et al, U.S. Dept. Agr. Cir. 400 (1936)), takes advantage of lactose fermentation and the use of a pH color indicator in an attempt to screen milk samples for the presence of Streptococcus aqalactiae (Bovine Mastitis, Schalm, O. W. et al, Lea Febiger, Philadelphia (1971), particularly pages 150-164). The purpose of the Hotis test is only to identify the presence of Streptococcus agalactiae in the cow.
The Hotis test is not applicable to the method of the present invention because it is not an antibiotic sensitivity test. Further, the Hotis test is disadvantageous because it is generally not accurate in clinical cases of mastitis (Bovine Mastitis, Schalm, O. W. et al, Lea Febiger, Philadelphia (1971), particularly pages 150-164).